1. Technical Field
The present disclosure relates to methods for the quantification of biological material in a liquefied sample, and to devices for partitioning and holding the biological material during quantification.
2. Discussion of Related Art
The determination and enumeration of microbial concentration is an essential part of microbiological analyses in many industries, including water, food, cosmetic, and pharmaceutical industries. For example, the determination of bacterial concentration in water is an essential part of water quality testing. In the United States, the USEPA regulations require that no Coliform or Escherichia coli (E. coli) can be present in a 100-ml potable water sample. The “presence/absence” format of a testing medium, such as COLILERT® and/or COLILERT-18® chemical mixtures (available from IDEXX Laboratories, Westbrook, Me.), either of which is used as a testing medium for detecting E. coli and coliform bacteria, is very useful in making this determination. Colilert® chemical mixture is based on the Defined Substrate Technology® described in Edberg, “Method and Medium for use in Detecting Target Microbes In Situ in A Specimen Sample of A Possibly Contaminated Material” See U.S. Pat. Nos. 4,925,789, 5,429,933, and 5,780,259.
However, there are areas where the quantification, not just the detection, of bacterial concentration is important. Examples of such areas include water waste, incoming water in water purification systems, surface water, recreational water and food testing.
The classical methods for the quantification of biological material are the membrane filtration (MF) technique and the multiple tube fermentation (MTF) technique using most probable number (MPN) methods. In membrane filtration, the required volume of sample is filtered through a membrane of a very small pore size to non-specifically trap bacteria. The membrane is then placed on a medium, which supports the growth of the target bacteria. The medium is then incubated at a specific temperature for a specific time, and any resulting colonies are counted. The MF technique is labor intensive and requires skilled microbiologists to perform the assay. In addition, a sample containing particles other than bacteria (e.g., wastewater or recreational water samples) may clog the membrane and make it unusable.
The MPN method for the MTF technique is described in Recles et al., “Most Probable Number Techniques” published in “Compendium of Methods for the Microbiological Examination of Foods”, 3rd ed. 1992, at pages 105-199, and in Greenberg et al., “Standard Methods For the Examination of Water and Wastewater” (8th ed. 1992). In this method, a volume of water sample is dispensed into several tubes (e.g., 10×10 tubes each containing 10 ml) and bacteria in each tube allowed to grow. After incubation at a specific temperature for a specific time, the number of positive tubes is counted. The microbiological quantification devices and methods using the MPN method are commercially available. Devices and Methods such as Quanti-Tray® and Quanti-Tray® 2000 (IDEXX Corporation, Westbrook, Me.) are used for bacterial quantification for drinking water, surface water, recreational water and wastewater samples. A detailed disclosure of these methods and devices is in Naqui et al. U.S. Pat. Nos. 5,518,892; 5,620,895; and 5,753,456. To perform these tests, the separate steps of adding the sample/reagent to the device and then sealing the device with a separate sealing apparatus are required before the incubation period. These methods and devices offer a significant improvement over the traditional MTF techniques in terms of their ease of use and also allow for accurate quantification of microorganisms in the sample. However, devices of this type may require the use of an instrument to distribute the sample/medium mixture into each individual compartment.